Group leader: Begoña Benito Casado - Associate Professor

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Na+ and K+ homeostasis in fungi and plants

“Unravelling the role of the ionic transporters involved in Na+ and K+ homeostasis may be an advance in the understanding of those that possibly are relevant effectors in the response to mineral nutrition deficiency and saline stress in plants and fungi”


Under the general objective of our group aimed at identifying and characterizing the main transporters involved in K+ mineral nutrition and Na+ transport in fungi and plants, two specific objectives have recently been developed:

- Characterization of molecular mechanisms involved in Na+ uptake in plant roots

- Identification of transporters involved in salt tolerance and in mineral K+ nutrition of plants


The progressive salinization of cultivated land affects over 800 million hectares (Rengasamy, 2010, Funct Plant Biol 37) and is one of the major concerns in agriculture. The processes that produce soil salinization may be complex based on climatic, soil, and groundwater conditions (Rengasamy, 2006 J Ex Bot 57) but the effects of salinity in crop plants are the same in all cases: water deficit and salt toxicity. Despite many efforts of research activity trying to characterize the Na+ transport and distribution along the plant, little is known about the molecular mechanisms involved in those processes. Besides, it is well accepted that a high cytoplasmic K+/Na+ ratio is a key determinant of plant salt tolerance meaning that plants with high Na+ accumulation in leaves are more salt sensitive. Until now our group has been interested in clarifying these aspects with two plant models: Arabidopsis and the bryophyte Physcomitrella patens. More recently, we have opened a new line of research considering that in nature plants live in symbiotic associations with multitude of soil microorganisms that can contribute/modify the plant response to different abiotic stresses. The plant-fungi model that we have chosen for this study is the symbiotic association between the endophyte Serendipita indica (formerly Piriformospora indica) and the plants Arabidopsis thaliana and rice.

Phenotypic approach on Arabidopsis to characterize the determinants involved in Na+ tolerance

We studied the natural variability of different accessions of Arabidopsis on Na+ tolerance considering the Na+ toxicity evidenced by “leaf wilting” or “short root length” as quantitative traits. The difference of this approach compared to others performed earlier is that the NaCl concentrations to which the plants were exposed were not too high to avoid superimposed osmotic effects. Our results indicate that Arabidopsis plants growing in NaCl can suffer root or shoot Na+ toxicities and an ionic growth inhibition, not due specifically to Na+ but also induced similarly by K+ (See Fig. 1)



Na+ uptake by plant roots and osmotic adjustment in saline conditions

We have studied the Na+ transport that operates in roots of Arabidopsis exposed to saline conditions and that constitutes an important pathway for the accumulation of Na+ in Arabidopsis shoots. This Na+ transport is mediated or controlled by one or more nitrate-dependent transporters. It implies an interesting result since it is shown that the Na+ uptake, considered in many cases as detrimental for plant, is coupled to nitrate uptake, an essential mineral plant nutrient. It suggests that Na+, at least at milimolar concentrations studied (up to 20-60 mM NaCl), has not a toxic but an essential role. Our work also indicates that other anions such as chloride may also function in the Na+ loading into the xylem and thus in its transfer to the shoots.

In order to find out which are the candidates to mediate the Na+ transport in plant roots, we carried out an Illumina RNA-Seq transcriptome profiling to identify those transporters that are differentially expressed in the moderate saline conditions tested, in the presence and absence of nitrate. Among the differentially expressed genes identified in any of the saline conditions, we have selected some gene encoding transporters as candidates to mediate Na+ transport for a further phenotypic analysis of their respective T-DNA mutants (See Fig. 2)



In addition, we also have seen in Arabidopsis that, under high osmolality conditions (400 mOsm), Na+ acts as an osmotic stabilizer in such a way that its uptake prevent plant water loss and shoot wilting. These results encourage us to rethink that Na+ entry by roots, besides being unavoidable, can be beneficial to the plant because it determines osmotic adjustments in plants growing in saline conditions. (See Fig. 3)



Contribution of chloroplast to salt tolerance in plants

It is assumed that the main strategy to maintain a low Na+ concentration in the cytosol is its compartmentation in the vacuole. However, using Corona Green, a Na+ binding indicator, we have observed that Na+ accumulates in chloroplasts of Physcomitrella and that this accumulation apparently does not affect the chloroplast function. These results suggest that chloroplast might have a relevant role to control cytosolic Na+ in saline conditions. We have carried out an ultra-structural analysis of Physcomitrella plants exposed to saline conditions and we have observed that in these conditions, protonema cells showed a retraction of plasma membrane from the cell wall, a compactation of the cytosol, an increase in vacuolization and specially, a striking increase in starch granules numbers accumulated in chloroplasts. These results would suggest that a higher starch accumulation in the NaCl-treated plants may be important for salt tolerance. Similar results has also been obtained in Thellungiella (Xuchu Wang et al., 2013 Mol Cell Proteom 12). (See Fig. 4).



In order to know whether the NHAD chloroplast transporters have any role in salt tolerance, we have carried out the cloning, expression and functional characterization of nhad knockout mutants in Physcomitrella. The results indicate that NHAD transporters have not any relevant role by modifying Na+ accumulation in the chloroplast, but probably in the regulation of osmotic or pH changes occurring under saline conditions.

Study of the Na+ and K+ homeostasis during the symbiotic associations of the endophyte Serendipita indica with Arabidopsis and rice plants

The most recent goal in which we are involved is in the identification of the Na+ and K+ transport systems that operate during the symbiosis between plants and microorganisms associated and to determine their contribution to the mineral nutrition and salt tolerance of plants growing in saline conditions. In a first attempt we have initiated the characterization of the ENA ATPases of the fungus. These are proteins involved in Na+ efflux from cells that have demonstrated to be very effective systems to increase salt tolerance in other fungi. Two genes, PiENA1 and PiENA5 have been identified in the Piriformospora genome (https://genome.jgi.doe.gov/Pirin1/Pirin1.home.html) and these are now under study. (See Fig.5).



Representative Publications

Lanza, M., Haro, R., Conchillo, L.B., Benito, B. 2019. The endophyte Serendipita indica reduces the sodium content of Arabidopsis plants exposed to salt stress: fungal ENA ATPases are expressed and regulated at high pH and during plant co-cultivation in salinity. Environmental Microbiology. DOI: 10.1111/1462-2920.14619

Lanza, M., Haro, R., Conchillo, L.B., Benito, B. 2019. The endophyte Serendipita indica reduces the sodium content of Arabidopsis plants exposed to salt stress: fungal ENA ATPases are expressed and regulated at high pH and during plant co-cultivation in salinity. Environmental Microbiology. DOI: 10.1111/1462-2920.14619

Álvarez-Aragón, R.; Rodríguez-Navarro, A. 2017. "Nitrate-dependent shoot sodium accumulation and osmotic functions of sodium in Arabidopsis under saline conditions". Plant Journal 91, 208–219. DOI: 10.1111/tpj.13556".

Ruiz-Lau, N; Sáez, Á; Lanza, M; Benito, B. 2017. "Genomic and transcriptomic compilation of chloroplast ionic transporters of Physcomitrella patens. Study of NHAD transporters in Na+ and K+ homeostasis". Plant and Cell Physiology. DOI: 10.1093/pcp/pcx150".

Álvarez-Aragón, R.; Haro, R.; Benito, B.; Rodríguez-Navarro, A. 2016. "Salt intolerance in Arabidopsis: shoot and root sodium toxicity, and inhibition by sodium-plus-potassium overaccumulation". Planta 243, 97-114. DOI: 10.1007/s00425-015-2400-7".

Dominguez-Nuñez, JA; Benito, B; Berrocal-Lobo, M; Albanesi, A. 2016. "Mycorrhizal Fungi: Role in the Solubilization of Potassium", p. 77-98. In S. V. Meena, R. B. Maurya, P. J. Verma, and S. R. Meena (eds.), Potassium Solubilizing Microorganisms for Sustainable Agriculture. Springer India, New Delhi. DOI: 10.1007/978-81-322-2776-2_6".

Ruiz-Lau, N; Bojórquez-Quintal, E; Benito, B; Echevarría-Machado, I; Sánchez-Cach, LA; Medina-Lara, MdF; Martínez-Estévez, M. 2016. "Molecular cloning and functional analysis of a Na+-insensitive K+ transporter of Capsicum chinense Jacq". Frontiers in Plant Science. DOI: 10.3389/fpls.2016.01980".

Nieves-Cordones, M; Martinez, V; Benito, B; Rubio, F. 2016. "Comparison between Arabidopsis and rice for main pathways of K+ and Na+ uptake by roots". Frontiers in Plant Science. DOI: 10.3389/fpls.2016.00992".

Benito, B; González-Guerrero, M. 2014. "Unravelling potassium nutrition in ectomycorrhizal associations". New Phytologist. DOI: 10.1111/nph.12659".

Benito, B; Haro, R; Amtmann, A; Cuin, TA; Dreyer, I. 2014. "The twins K+ and Na+ in plants". Journal of Plant Physiology. DOI: 10.1016/j.jplph.2013.10.014".





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